TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles
复制标题
DOI：
10.1186/s12974-019-1529-x
复制
发表时间：
2019-07-04
影响因子：
9.3
通讯作者：
Pascual, Maria
中科院分区：
文献类型：
--
作者：
Ibanez, Francesc;Montesinos, Jorge;Pascual, Maria
研究背景细胞外囊泡（extracellular vesicles，EVs）参与细胞间信号传导，参与神经炎症的调节和放大。我们以前已经表明，乙醇激活胶质细胞通过Toll样受体4（TLR 4）通过触发神经炎症。在这里，我们评估，如果乙醇和TLR 4反应改变释放和炎性含量的星形胶质细胞衍生的EV，以及这些囊泡是否能够通过传播neuroniflammation. MethodsCortex神经元和星形胶质细胞在文化中进行沟通。从用或不用乙醇（40 mM）处理24小时的WT和TLR 4-KO星形胶质细胞的原代培养物的细胞外培养基中分离EV。采用流式细胞术、纳米颗粒跟踪分析技术、外泌体分子标记物（四跨膜蛋白）沿着电镜技术对EV进行表征和定量。通过Western blot和RT-PCR分析星形胶质细胞来源的EV和与这些EV一起孵育或不孵育的神经元中EV在炎性蛋白、mRNA和miRNA中的含量。功能分析的miRNAs也performed.ResultsWe表明，乙醇增加分泌的nanovesicles和它们的内容，通过提高炎症相关蛋白（TLR 4，NF κ B-p65，IL-1 R，caspase-1，NLRP 3）的水平，并通过改变微RNA（mir-146 a，mir-182，和mir-200 b）的EV从WT-星形胶质细胞相比，那些从未经处理的WT细胞。在未处理的和乙醇处理的TLR 4-KO星形胶质细胞之间，未观察到分离的EV的数量或其含量的变化。我们还表明星形胶质细胞衍生的EV可以被幼稚皮质神经元内化，以增加炎性蛋白（考克斯-2）和miRNA（例如，mir-146 a）并危及其生存。miRNAs的功能分析揭示了所表达的miRNAs在参与几个inflammatorypathways.ConclusionsThese结果表明，星形胶质细胞衍生的EVs可以作为炎症信号的细胞递质，通过扩散和放大由乙醇诱导的神经炎症反应，通过TLR 4激活。
BackgroundCurrent evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation.MethodsCortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40mM) for 24h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed.ResultsWe show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NF kappa B-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naive cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways.ConclusionsThese results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.